Previous results suggest that Ehrlich ascites tumor cell NaK-ATPase, but not NaK-ATPase isolated from normal cells, is phosphorylated on the enzyme's beta subunits. As part of our effort to determine if this observation indicates a protein kinase-\and protein phosphatase-dependent phosphorylation/dephosphorylation regulatory cycle of NaK-ATPase action, we have used column chromatofocusing to resolve two soluble alkaline phosphatase enzymes from the post-microsomal supernatant fractions of Ehrlich ascites tumor cells. Enzyme I has a pI of 5.6, and enzyme II has a pI of 4.9. This result is consistent with our observation that partially purified Ehrlich ascites tumor cell alkaline phosphatase samples subjected to native gel electrophoresis migrate at the level of the tracking dye and are thus highly negatively charged. Under our current assay conditions, using p-nitrophenylphosphate as substrate, the highest specific activity obtained for either one of these two enzymes is considerably less than 0.1 micromoles of substrate hydrolyzed/min/mg of protein. If these alkaline phosphatases show substrate specificity for Ehrlich ascites tumor cell NaK-ATPase, then their low activity could account for the fact that the NaK-ATPase is isolated in a phosphorylated form. In addition, we have made progress on the resolution of the control enzyme in this study, NaKATPase from mouse brain. The specific activity of this enzyme is approximately 10 micromoles of ATP hydrolyzed/min/mg of protein. We estimate the purity to be about 33% at this stage. Furthermore, other work has identified a potent inhibitor of NaK-ATPase in Ehrlich ascites tumor cell fluid. The inhibitor has a molecular weight greater than phosphate but less than that of ATP. It does not contain phosphate, however. Further work is in progress to identify the inhibitory compound and to determine if this is the elusive endogenous inhibitor of NaK-ATPase.